Bulletin OEPP/EPPO Bulletin (2013) 43 (3), 417–424 ISSN 0250-8052. DOI: 10.1111/epp.12059

European and Mediterranean Plant Protection Organization Organisation Europe´enne et Me´diterrane´enne pour la Protection des Plantes PM 7/115 (1)

Diagnostics Diagnostic

PM 7/115 (1) Drosophila suzukii

Specific scope Specific approval and amendment This standard describes a diagnostic protocol for Drosophila Approved in 2013-09. suzukii1.

Introduction Detection Drosophila suzukii (spotted wing drosophila) is a polypha- Drosophila suzukii may be detected as larvae or pupae in gous pest of many small fruits, especially from the genera fruits. Eggs may also be detected but are more difficult to Vaccinium, Rubus, Prunus, Fragaria, Vitis, Ficus, Actinidia, see. Adults can be caught in traps. Rhamnus, Lonicera, Sambucus and also many others. Plant species from 15 families are recorded as host plants Detection of adults (Kanzawa, 1939; Grassi et al., 2012; Seljak, 2011; Walsh et al., 2011). Drosophila suzukii is a vinegar fly. Most Different traps can be used: the most efficient method for – species of vinegar flies are not pests because they infest first detection is by trapping. Any 250 750 mL plastic con- overripe, fallen, rotting fruit. However, D. suzukii females tainers with closely fitting lids can be used as traps. Four to lay eggs in ripening fruit and larvae develop in the fruit six holes should be made on the side to enable flies to enter and cause it to become soft and unmarketable. It originates through these holes. To make the trap more selective and and is widely distributed in the temperate East Palaearctic to avoid the trapping of large insects the diameter of these zoogeographical region, but recently D. suzukii has been holes should not be too large. The recommended diameter introduced and is now reported from North America as well is about 3 mm. Various baits are used to attract the flies to as from Europe (Hauser, 2011; Cini et al., 2012). The most the trap. Apple cider vinegar is considered to be highly recent and comprehensive overview on introduction, spread, effective, and the most practical bait to use (EPPO, 2010). distribution, host range, economic impact and control in In trials, a mixture of vinegar and wine trapped signifi- Europe has been provided by Cini and collaborators (Cini cantly more D. suzukii compared with vinegar alone or et al., 2012). The pest is currently spreading quickly in wine alone. These results indicate a synergy of acetic acid Europe. It can be found at different altitudes from coast to and ethanol as lures for D. suzukii (Landolt et al., 2012). mountain. Because of the rapid spread, it is recommended Several other feed attractants and lures are proposed to be to consult PQR (EPPO, 2013) for updated information on effective in trapping D. suzukii. the geographical distribution. Identification Identity Morphological identification to species level with a binocu- lar microscope is the recommended diagnostic method. Name: Drosophila suzukii (Matsumura 1931) Magnification 910 for males to 950 for females, 9200 for Synomym: Leucophenga suzukii Matsumura 1931. male and female genitalia as well as for male sex combs Taxonomic position: Diptera, Brachycera: Drosophilidae; (if needed, guidance for the preparation of genitalia and Drosophila, subgen.: Sophophora. male sex combs is given in Appendix 1). EPPO code: DROSSU. A reliable morphological identification can only be per- Phytosanitary categorization: EPPO A2 List no. 363; EU formed on adult specimens. It is not possible to identify not categorized. with certainty eggs, larvae and pupae with this protocol (these should be reared to the adult stage). Immature stages 1Use of brand names of chemicals or equipment in these EPPO Stan- can only be identified by molecular techniques (Hauser, dards implies no approval of them as others may also be suitable.

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2011) however, there is currently little experience regarding molecular tests in the EPPO region. It should be noted that rearing adults from larvae is easy and rapid and morpholog- ical identification rather straightforward. An EPPO Standard PM 7/XX on DNA barcoding as an identification tool for plant pests is in preparation and COI sequences are cur- rently available in Q-bank for this pest. However, sequence data for other members of the suzukii-subgroup are not yet available and therefore, it is unclear whether a barcode-gap exists for D. suzukii. An excellent comparative diagnostic account on D. suzukii has been published by Hauser (Hauser, 2011). A good key for the identification has also been provided by Vlach (Vlach, 2012).

Fig. 1 Drosophila suzukii – male. Description of adults General Adults of D. suzukii are small flies approximately 2.25– 4.0 mm long with a wing span of 6–8 mm. (Figs 1,2 and 3) Males are usually slightly smaller (2.0–3.5 mm) than females (2.5–4.0 mm) (Kanzawa, 1939).

Head Head entirely yellow; compound eyes red to red orange in live specimens, covered with short and dense pilosity, gena about 0.3 times as high as eye, facial ridge with two sub- equal vibrissal setae (Fig. 4), palpi with only one prominent seta at tip; antennae 3-segmented all segments yellow, termi- nal segment (flagellum) with a dorsal plumose arista; arista typically forked at the tip through the main stem and the last lateral hair, which is one of the key characters in separating Drosophilidae from other Diptera families with plumose arista (Fig. 4); frons with 3 pairs of orbital setae (the most Fig. 2 Drosophila suzukii – male. anterior one proclinate, the remaining two reclinate); ocellar setae present, inserted inside the ocellar triangle; a pair of postocellar setae well developed, convergent (Fig. 5).

Thorax Dorsum yellowish brown, sometimes with two darker brown longitudinal stripes in the dorsocentral area that sometimes may be fused into one; 8 rows of acrostichal setulae between the anterior pair of dorsocentral setae, 2 subequal humeral setae (Fig. 5). Wings 2.3–3.3 mm, entirely hyaline in females, while in males a typical large black spot at the top of vein R2+3 is present (Figs 1,2 and 6) by which D. suzukii differs from all indigenous European Drosophila species (note, however, that many East-Palae- arctic and Oriental Drosophila species belonging to the suzukii subgroup may exhibit similar feature); sometimes this spot may be fade or even absent in still teneral speci- mens. Very seldom mature “spotless” male individuals may Fig. 3 Drosophila suzukii – female. also occur. In these cases, other morphological characters such as sex combs and genital segment have to be exam- present; forelegs in males with two sex combs, one on the ined as well. Halterae are white to bright yellow. Sternum first and one on the second tarsomere, both in one row only and legs yellow. Legs: preapical dorsal seta on all tibia and oriented parallel to the length of the tarsus, the first

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Fig. 7 Drosophila suzukii - sex combs on first and second tarsomere of male’s forelegs. Fig. 4 Drosophila suzukii - head (frontal view). Male. Male’s genital segment see (Fig. 10); epandrium slightly constricted at middle; surstylus (clasper) large, with tapering tip, bearing 10–11 primary teeth (prensisetae) medi- ally and 4–5 secondary teeth subapicaly; marginal bristles also in two sets, upper one 5–6, lower one 3–4 at the tip; anal plate oval, tapered ventrally (adopted after Hsu, 1949).

Females. Females have a strongly sclerotized ovipositor with strong, contrastingly black marginal teeth by which females of D. suzukii are easy distinguishable from all other European as well as North American Drosophila spe- cies; each ovipositor valve bears 30–36 teeth, whereby those in distal half of each valve are much stronger and darker than proximal ones (Fig. 11). The ovipositor is 6–7 times as long as the diameter of the spermatheca, while in most European species the ovipositor is only 2–4 times as Fig. 5 Drosophila suzukii - the basic chaetotaxy of head and notum long as the spermatheca (Hauser, 2011). Similar strong (acr s - acrostichal setae (8 rows), dc s - dorsocentral setae, oc s - sclerotized ovipositors are exhibited by some other East- ocellar setae, orb s - orbital setae, poc s - postocellar setae, pprn s - Palaearctic and Oriental species of the suzukii subgroup, postpronotal setae). such as Drosophila subpulchrella Takamori & Watabe, Drosophila pulchrella Tan, Hsu & Sheng, Drosophila immacularis Okada (Okada, 1966:81–89; Takamori et al., 2006; Kimura & Anfora, 2011).

Egg Eggs are milky white and glossy, semi-transparent, on aver- age 0.62 9 0.18 mm wide (Fig. 12). The eggs have two subapical respiratory tubes (Kanzawa, 1939).

Larva There are three larval instars that range in size (length 9 width) from 0.67 9 0.17 mm, 2.13 9 0.40 mm Fig. 6 Drosophila suzukii - right wing of a male with indicated veins. and 3.94 9 0.88 mm on average for first, second and third instars, respectively (Kanzawa, 1939) (Fig. 13). The larvae one composed of 5–6 and the second one of 3–4 strong are white to cream in colour with visible internal organs and spines (Fig. 7); females lack such sex combs. black cephalopharyngeal skeleton; mandibles (mouthhooks) of the third instar larva densely serrated ventrally; each ante- Abdomen rior spiracle composed of 6 to 8 subparallel respiratory Abdominal segments with large dark brown to black trans- tubules; posterior spiracles horn-shaped, both tubes parallel, versal bands that are not broken or interrupted medially close to each other; sternites with transversal rows of brown (Fig. 8; compare also to Fig. 9). rose-thorn-shaped sharp spinules.

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Fig. 8 Drosophila suzukii - abdominal tergits with transversal medially not interrupted dark bands (arrows).

Fig. 11 Drosophila suzukii - female with strongly sclerotized saw-like ovipositor.

Fig. 9 Drosophila immigrans - abdominal tergits with transversal medially interrupted (arrows!) dark bands on segments 2–5.

Fig. 12 Drosophila suzukii – eggs.

on both sides of the head. Each respiratory tube bears seven to eight radially arranged branches at the ends (Kanzawa, 1935).

Related and morphologically similar species that may be confused with D. suzukii The conspicuous black spots on the wing top near the vein

R2+3 (Fig. 6) and the two sets of tarsal sex combs in males, as well as strongly sclerotized and serrated ovipositor in females make the identification of D. suzukii in Europe fairly easy. No one indigenous Drosophila species exhibit similar characters. In the East-Palaearctic and Oriental zoo- geographical region (India, South East Asia, Southern Fig. 10 Drosophila suzukii - genital segment of a male (flattened China), however, several close related species with similar view): ap - anal plate, ep - epandrium, pt - primary teeth, ss - surstylus, appearance and behaviour occur (Kimura & Anfora, 2011). st - secondary teeth. Nearly identical wing spotting in males are show by D. subpulchrella, recently described from Japan and China Pupa (Takamori et al., 2006) and Drosophila biarmipes known The pupae of Drosophila suzukii are creamy becoming tan– from India (Malloch, 1924). Another similar species is brown in colour and are about 3 mm long by 1 mm wide D. pulchrella, but it has an additional much smaller black (Fig. 14). The horn-shaped respiratory tubes are about spot on the wing base as well (Okada, 1966). Males of all 0.3 mm long and are protrusions of the anterior spiracles three species differ from D. suzukii by the shape of sex

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Fig. 13 Drosophila suzukii - third instar larva. Fig. 16 Drosophila melanogaster - a sex comb on first tarsomere of male’s forelegs.

Fig. 14 Drosophila suzukii – pupae.

Fig. 17 Drosophila immigrans - ovipositor moderately sclerotized with subequal coloured teeth on valve margins.

Fig. 15 Drosophila suzukii - ovipositor with strong black teeth on valve margins.

Fig. 18 Drosophila melanogaster - soft ovipositor with weak teeth; combs on the first and the second tarsomere. In D. suzukii spermatheca (arrow). they are composed of a single row of spines on both the first and second tarsomere, while in the other three species (Okada, 1966; Bock & Wheeler, 1972). In D. biarmipes, sex comb spines are arranged in two separate rows (upper the sex comb is only present on the first tarsomere and and lower) on each tarsomere or at least on the first one female’s ovipositor is weakly sclerotized. As in D. suzukii,

ª 2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 417–424 422 Diagnostics strongly sclerotized ovipositors with dark stout teeth are and acrostichal hairs are arranged in six rows. Good also present in the females of D. subpulchrella, D. pulchrella keys for separation of D. suzukii from D. pulchrella and and D. immacularis (Kimura & Anfora, 2011). The number D. pulchrella from D. immacularis were provided by of teeth on valvae margins, however, is lower (usually 20 Okada (Okada, 1956, 1966). All species with long and to 25 in number) in these three species. Furthermore, upper strongly sclerotized ovipositors (D. suzukii, D. subpulchrella, marginal teeth (usually three in number) are as strong D. pulchrella and D. immacularis) have putative similar as lower marginal teeth, while they are markedly smaller ecology laying eggs in fresh fruits (Kimura & Anfora, in D. suzukii (Fig. 15). In D. immacularis, the black spot 2011). at the top of first vein is always absent in both sexes

Dichotomous key for identification of Drosophila suzukii (Matsumura) This key only works properly when all characters are considered together step by step. Single characters cannot be taken from the key and used independently as this could lead to misidentification.

1 Small sized flies of <4 mm body length and golden yellow to yellowish brown colours (Figs 1,2 2 and 3); head with red or red orange, short pilose compound eyes (observe at slant or backlight), 3 segmented antennae, flagellum (3rd segment) with a plumose arista characteristically forked at the tip (Fig. 4), 3 pairs of orbital setae (one proclinate, 2 reclinate), a pair of convergent postocellar setae present (Fig. 5), vibrissae always present (Fig. 4); wing as in (Fig. 6), subcosta (Sc) incomplete, not reaching costal margin, humeral and subcostal break well developed (Drosophilidae p. p.) 1* Disagreeing with above in one or more of characters Not Drosophila

2 Wings transparent (Fig. 3) or with a single dark spot near the end of wing on first vein R2+3 3 (Figs 1, 2 and 6) 2* Wings with bands, multiple spots, with a darkened crossvein, or a spot at the tip of the wing Not D. suzukii

on the second vein (R4+5) 3 Abdominal tergites 2 to 5 with broad dark brown transversal bands NOT broken or interrupted 4 in mid-dorsal line (Drosophila subgenus Sophophora) (Fig. 8, compare also Fig. 9) 3* Abdominal tergites with stripes, spots, or patterns other than above or completely black; if Not D. suzukii transversal bands developed they are broken or interrupted medially by a longitudinal light coloured strip at least on tergites 2 to 5 (Fig. 9)

4 Wing with a single black spot near wing tip at the junction of first vein (R2+3) with the costal 5 margin (Fig. 6); forelegs with sex comb(s) on tarsomeres 4* Without such a spot near wing tip (Fig. 3); with or without sex combs 6 5 Forelegs with two sex combs, one on 1st tarsomere composed of 5–6 spines and one on 2nd D. suzukii (male) tarsomere composed of 3–4 spines respectively the spines of both oriented Æ parallel to the length of the tarsus (Fig. 7); genital segment as in (Fig. 10) 5* Forelegs with one (e.g. Fig. 16) or several sex combs on tarsomeres; if two sex combs then Some other Drosophila species their spines directed more or less transversally or obliquely to the length of the tarsus and/or from the suzukii-subgroup sex comb spines arranged in two or more transverse rows on each tarsomere (species not occurring yet in Europe) 6 Females: ovipositor developed, forelegs without sex combs on tarsomeres 7 6* Males: forelegs with one or several sex combs on tarsomeres (Figs 7 and 16) 10 7 Large and strong sclerotized ovipositor with strong teeth (Figs 11, 15 and 17) 8 7* Ovipositor not sclerotized, soft with weak teeth (Fig. 18) Other Drosophila 8 Marginal teeth on ovipositor strong, almost black, much darker than ovipositor itself (Figs 11 and 15) 9 8* Teeth on ovipositor the same colour or only slightly darker as the rest of ovipositor (Fig. 17) Other Drosophila, not D. suzukii 9 Ovipositor saw-like, with strong contrastingly black teeth in the distal half of each valve; 30–36 in D. suzukii (female) number on each valve; teeth on upper margin (usually 3 in number) much weaker than distal lower teeth (Figs 11 and 15) 9* Ovipositor arched; number of teeth lower, usually <25, teeth on upper margin as stout as that on lower D. subpulchrella, D. pulchrella, margin (species not occurring yet in Europe) D. immacularis 10 Forelegs with two sex combs, one on 1st tarsomere composed of 5–6 spines and one on 2nd tarsomere D. suzukii (“spotless” and teneral composed of 3–4 spines respectively, the spines of both oriented Æ parallel to the length of the tarsus males) (Fig. 7); genital segment as in (Fig. 10) 10* Forelegs with one or several sex combs on tarsomeres; if two then their spines directed more or less Males of other Drosophila, transversally or obliquely to the length of the tarsus or sex comb spines in more than one row on each but not D. suzukii tarsomere (e.g. Fig. 16)

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Reference material Drosophila suzukii, available at: http://www.eppo.int/QUARANTINE/ Alert_List/insects/Drosophila_suzukii_factsheet_12-2010.pdf (last acc- Material can be obtained from laboratories which have essed on 1 Sept 2013). declared expertise on this pest in the EPPO Database on EPPO (2010) EPPO Datasheet on Drosophila suzukii, available at http:// Diagnostic expertise. www.eppo.int/QUARANTINE/Alert_List/insects/Drosophila_suzukii_ factsheet_12-2010.pdf (last accessed on 01 Sept 2013). EPPO (2013) PQR - EPPO database on quarantine pests (available Reporting and documentation online). http://www.eppo.int [accessed on 1 Sept 2013]. Grassi A, Palmieri L & Giongo L (2012) Drosophila (Sophophora) Guidelines on reporting and documentation are given in suzukii (Matsumura), new pest of soft fruits in Trentino (North-Italy) EPPO Standard PM7/77 (1) Documentation and reporting and in Europe. IOBC/wprs Bulletin 70, 121–128. on a diagnosis. Hauser M (2011) A historic account of the invasion of Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) in the continental United states, with remarks on their identification. Pest Management Further information Science, 67, 1352–1357. Hsu TC (1949). The external genital apparatus of male Drosophilidae Further information on this organism can be obtained from: in relation to systematics. The University of Texas Publications, G. Seljak, Agricultural and Forestry Chamber of Slove- 4920(XI): 80–142. nia, Pri hrastu, 18 SI-5000, Nova Gorica, Slovenia gabrijel. Kanzawa T (1935). Research into the Fruit-fly Drosophila suzukii [email protected]. Matsumura (Preliminary Report). Yamanashi Prefecture Agricultural Experiment Station Report. M. Hauser Senior Insect Biosystematist California Kanzawa T (1939). Studies on Drosophila suzukii Mats. Kofu, Department of Food and Agriculture Plant Pest Diagnostics Yamanashi agricultural experiment station 49 pp. Abstract in Review Branch 3294 Meadowview Road Sacramento, CA 95832- of Applied Entomology, 29: 622. 1448 [email protected]. Kimura MT & Anfora G (2011). Evolution and Ecology of Drosophila suzukii: a comparison between native and invaded areas. International meeting: Drosophila suzukii: new threat for European fruit production, Feedback on this diagnostic protocol Trento, December 2, 2011. PDF presentation available on: http://cri. fmach.eu/publications/report/Drosophila-talks [accessed on 1 Sept 2013]. If you have any feedback concerning this Diagnostic Pro- Malloch JR (1924) Two Drosophilidae from Coimbatore. Memoirs of tocol, or any of the tests included, or if you can provide the Department of Agriculture in India 6,63–65. additional validation data for tests included in this proto- Landolt PJ, Adams T & Rogg H (2012) Trapping spotted wing col that you wish to share please contact diagnos- drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), [email protected]. with combinations of vinegar and wine, and acetic acid and ethanol. Journal of Applied Entomology, 136, 148–154. Okada T (1956). Systematic Study of Drosophilidae and Allied Families Protocol revision of Japan. pp. 283. Gihodo, Tokyo, Japan. Okada T (1966). Diptera from Nepal: Cryptochaetidae, Diastetidae & An annual review process is in place to identify the need Drosophilidae. Bulletin of the British Museum (natural history). for revision of diagnostic protocols. Protocols identified Entomology (Suppl. 6) 129. as needing revision are marked as such on the EPPO Seljak G (2011). Drosophila suzukii (Matsumura) in Slovenia: current website. knowledge on its distribution and phytosanitary impact. International When errata and corrigenda are in press, this will also be meeting: Drosophila suzukii: new threat for European fruit production. Trento (Italy), 2.12.2011. PDF presentation available by marked on the website. clicking on the title Drosophila suzukii (Matsumura) in Slovenia on the following page in the Google browser https://www.google.si/ Acknowledgements search?q=Seljak+Drosophila+suzukii+Trento&oq=Seljak+Drosophila+ suzukii+Trento&aqs=chrome..69i57.21099j0j8&sourceid=chrome& This protocol was originally drafted by: G. Seljak, Agricul- espv=210&es_sm=122&ie=UTF-8 [accessed 23 Sept 2013]. tural and Forestry Chamber of Slovenia. Takamori H, Watabe H, Fuyama Y, Zhang Y & Aotsuka T (2006) Drosophila subpulchrella, a new species of the Drosophila suzukii species subgroup from Japan and China (Diptera: Drosophilidae). References Entomological Science, 9, 121–128. Vlach J (2012) Identifying Drosophila suzukii. [Online available]: Bock IR & Wheeler MR (1972). I. The Drosophila melanogaster http://www.agf.gov.bc.ca/cropprot/swd_identification.pdf [accessed on Species Group. The University of Texas Publication 72/3; 1–102. 1 Sept 2013]. Cini A, Ioriatti C & Anfora G (2012). A review of the invasion Walsh DB, Bolda MP, Goodhue RE, Dreves AJ, Lee JC, Bruck DJ of Drosophila suzukii in Europe and a draft research agenda et al. (2011) Drosophila suzukii (Diptera: Drosophilidae): Invasive for integrated pest management. Bulletin of Insectology 65: 149– pest of ripening soft fruit expanding its geographic range and 160. damage potential. Journal of Integrated Pest Management, 1,1–7.

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Appendix 1 – Examination of genitalia and in glacial acetic acid at room temperature for about 15 min male sex combs and then to absolute ethanol for several minutes. Gently For more accurate examination of genitalia, preparation of separate the genital segment or the last segments of abdo- microscope slides is recommended. First gently remove the men. Further procedures depend on the type of preparation. abdomen and place it into absolute ethanol, to which an For routine examination temporary slides are usually per- equal volume of approximately 10% KOH is added. Macer- formed. For temporary mounts the simplest technique is to ate the abdomen in this mixture at room temperature for place the dissected parts in a drop of glycerol on a micro- about 12–24 h. This procedure can be accelerate by heating scope slide and cover then carefully with a coverslip over on a hot plate at about 90–95°C for 5–10 min. Gently it. For permanent slides Canada balsam, Berlese fluid, squeeze abdomen with a small spatula or other tool to Euparal or some other mountants can be used. remove the macerated content. Transfer then the abdomen

ª 2013 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 43, 417–424